Cranial modularity shifts during mammalian evolution

The mammalian skull has been studied as several separate functional components for decades, but the study of modularity is a more recent, integrative approach toward quantitative examination of independent subsets of highly correlated traits, or modules. Although most studies of modularity focus on developmental and genetic systems, phenotypic modules have been noted in many diverse morphological structures. However, few studies have provided empirical data for comparing modules across higher taxonomic levels, limiting the ability to assess the broader evolutionary significance of modularity. This study uses 18-32 three-dimensional cranial landmarks to analyze phenotypic modularity in 106 mammalian species and demonstrates that cranial modularity is generally conserved in the evolution of therian mammals (marsupials and placentals) but differs between therians and monotremes, the two extant subclasses of Mammalia. Within therians, cluster analyses identify six distinct modules, but only three modules display significant integration in all species. Monotremes display only two highly integrated modules. Specific hypotheses of functional and developmental influences on cranial bones were tested. Theoretical correlation matrices for bones were constructed on the basis of shared function, tissue origin, or mode of ossification, and all three of these models are significantly correlated with observed correlation matrices for the mammalian cranium.     

The Developmental Basis of Variational Modularity: Insights from Quantitative Genetics,Morphometrics, and Developmental Biology

Groups of correlated characters (variational modules) often are considered to be the result of dissociated local developmental factors, i.e., of a modular genotype–phenotype map. But certain sets of pleiotropic factors can equally well induce modular phenotypic variation—no local developmental factors are necessary for a modular covariance structure. It is thus not possible to infer genetic or developmental modularity from standing variation alone. Yet, only for approximately linear genotype–phenotype maps is the induced covariance structure stable over changes of the phenotypic mean. For larger genetic and phenotypic variation, such as on a macroevolutionary level, developmental effects often are nonlinear and variational modularity remains stable only when it is realized by local dissociated developmental factors with no overlap of pleiotropic ranges. The evo-devo concept of modularity concurs only at this macroevolutionary level with the quantitative notion of variational modularity. Empirical evidence on the genetic and developmental architecture underlying phenotypic variation is inconclusive and partly subject to methodological problems. Many studies seem to indicate modularized phenotypic variation and local clusters of QTL effects, whereas other studies find support for several alternative models of modularity and report continuous distributions of QTL effects. This inconsistency partly results from the neglect of spatial relationships among the measured traits. Given the complex development of higher organisms, a combination of pleiotropic factors and more local developmental effects with a hierarchical, overlapping, and more or less continuous distribution appears most likely.     

Modular genetic architecture of floral morphology in Nicotiana: quantitative genetic and comparative phenotypic approaches to floral integration

Animal‐pollinated flowers are complex structures that may require a precise configuration of floral organs for proper function. As such, they represent an excellent system with which we can examine the role of phenotypic integration and modularity in morphological evolution. We use complementary quantitative genetic and comparative phenotypic approaches to examine correlations among floral characters in Nicotiana alata, N. forgetiana and their artificial fourth‐generation hybrids. Flowers of both species share basic patterns of genetic and phenotypic correlations characterized by at least two integrated character suites that are relatively independent of each other and are not disrupted by four generations of recombination in hybrids. We conclude that these integrated character suites represent phenotypic modules that are the product of a modular genetic architecture. Intrafloral modularity may have been critical for rapid specialization of these species to different pollinators.     

Evolution of a complex phenotype with biphasic ontogeny: Contribution of development versus function and climatic variation to skull modularity in toads

The theory of morphological integration and modularity predicts that if functional correlations among traits are relevant to mean population fitness, the genetic basis of development will be molded by stabilizing selection to match functional patterns. Yet, how much functional interactions actually shape the fitness landscape is still an open question. We used the anuran skull as a model of a complex phenotype for which we can separate developmental and functional modularity. We hypothesized that functional modularity associated to functional demands of the adult skull would overcome developmental modularity associated to bone origin at the larval phase because metamorphosis would erase the developmental signal. We tested this hypothesis in toad species of the Rhinella granulosa complex using species phenotypic correlation pattern (P‐matrices). Given that the toad species are distributed in very distinct habitats and the skull has important functions related to climatic conditions, we also hypothesized that differences in skull trait covariance pattern are associated to differences in climatic variables among species. Functional and hormonal‐regulated modules are more conspicuous than developmental modules only when size variation is retained on species P‐matrices. Without size variation, there is a clear modularity signal of developmental units, but most species have the functional model as the best supported by empirical data without allometric size variation. Closely related toad species have more similar climatic niches and P‐matrices than distantly related species, suggesting phylogenetic niche conservatism. We infer that the modularity signal due to embryonic origin of bones, which happens early in ontogeny, is blurred by the process of growth that occurs later in ontogeny. We suggest that the species differing in the preferred modularity model have different demands on the orbital functional unit and that species contrasting in climate are subjected to divergent patterns of natural selection associated to neurocranial allometry and T3 hormone regulation.     

Cranial modularity and sequence heterochrony in mammals

Heterochrony, the temporal shifting of developmental events relative to each other, requires a degree of autonomy among those processes or structures. Modularity, the division of larger structures or processes into autonomous sets of internally integrated units, is often discussed in relation to the concept of heterochrony. However, the relationship between the developmental modules derived from studies of heterochrony and evolutionary modules, which should be of adaptive importance and relate to the genotype-phenotype map, has not been explicitly studied. I analyzed a series of sectioned and whole cleared-and-stained embryological and neonatal specimens, supplemented with published ontogenetic data, to test the hypothesis that bones within the same phenotypic modules, as determined by morphometric analysis, are developmentally integrated and will display coordinated heterochronic shifts across taxa. Modularity was analyzed in cranial bone ossification sequences of 12 therian mammals. A dataset of 12-18 developmental events was used to assess if modularity in developmental sequences corresponds to six phenotypic modules, derived from a recent morphometric analysis of cranial modularity in mammals. Kendall's tau was used to measure rank correlations, with randomization tests for significance. If modularity in developmental sequences corresponds to observed phenotypic modules, bones within a single phenotypic module should show integration of developmental timing, maintaining the same timing of ossification relative to each other, despite differences in overall ossification sequences across taxa. Analyses did not find any significant conservation of developmental timing within the six phenotypic modules, meaning that bones that are highly integrated in adult morphology are not significantly integrated in developmental timing.     

A statistical framework for testing modularity in multidimensional data

Modular variation of multivariate traits results from modular distribution of effects of genetic and epigenetic interactions among those traits. However, statistical methods rarely detect truly modular patterns, possibly because the processes that generate intramodular associations may overlap spatially. Methodologically, this overlap may cause multiple patterns of modularity to be equally consistent with observed covariances. To deal with this indeterminacy, the present study outlines a framework for testing a priori hypotheses of modularity in which putative modules are mathematically represented as multidimensional subspaces embedded in the data. Model expectations are computed by subdividing the data into arrays of variables, and intermodular interactions are represented by overlapping arrays. Covariance structures are thus modeled as the outcome of complex and nonorthogonal intermodular interactions. This approach is demonstrated by analyzing mandibular modularity in nine rodent species. A total of 620 models are fit to each species, and the most strongly supported are heuristically modified to improve their fit. Five modules common to all species are identified, which approximately map to the developmental modules of the mandible. Within species, these modules are embedded within larger "super-modules," suggesting that these conserved modules act as building blocks from which covariation patterns are built.     

Studying morphological integration and modularity at multiple levels: concepts and analysis

Although most studies on integration and modularity have focused on variation among individuals within populations or species, this is not the only level of variation for which integration and modularity exist. Multiple levels of biological variation originate from distinct sources: genetic variation, phenotypic plasticity resulting from environmental heterogeneity, fluctuating asymmetry from random developmental variation and, at the interpopulation or interspecific levels, evolutionary change. The processes that produce variation at all these levels can impart integration or modularity on the covariance structure among morphological traits. In turn, studies of the patterns of integration and modularity can inform about the underlying processes. In particular, the methods of geometric morphometrics offer many advantages for such studies because they can characterize the patterns of morphological variation in great detail and maintain the anatomical context of the structures under study. This paper reviews biological concepts and analytical methods for characterizing patterns of variation and for comparing across levels. Because research comparing patterns across level has only just begun, there are relatively few results, generalizations are difficult and many biological and statistical questions remain unanswered. Nevertheless, it is clear that research using this approach can take advantage of an abundance of new possibilities that are so far largely unexplored.     

The evolutionary role of modularity and integration in the hominoid cranium

Patterns of morphological integration and modularity among shape features emerge from genetic and developmental factors with varying pleiotropic effects. Factors or processes affecting morphology only locally may respond to selection more easily than common factors that may lead to deleterious side effects and hence are expected to be more conserved. We briefly review evidence for such global factors in primate cranial development as well as for local factors constrained to either the face or the neurocranium. In a sample comprising 157 crania of Homo sapiens, Pan troglodytes, and Gorilla gorilla, we statistically estimated common and local factors of shape variation from Procrustes coordinates of 347 landmarks and semilandmarks. Common factors with pleiotropic effects on both the face and the neurocranium account for a large amount of shape variation, but mainly by extension or truncation of otherwise conserved developmental pathways. Local factors (modular shape characteristics) have more degrees of freedom for evolutionary change than mere ontogenetic scaling. Cranial shape is similarly integrated during development in all three species, but human evolution involves dissociation among several characteristics. The dissociation has probably been achieved by evolutionary alterations and by the novel emergence of local factors affecting characteristics that are controlled at the same time by the common factors.     

The Influence of Modularity on Cranial Morphological Disparity in Carnivora and Primates (Mammalia)

Background

Although variation provides the raw material for natural selection and evolution, few empirical data exist about the factors controlling morphological variation. Because developmental constraints on variation are expected to act by influencing trait correlations, studies of modularity offer promising approaches that quantify and summarize patterns of trait relationships. Modules, highly-correlated and semi-autonomous sets of traits, are observed at many levels of biological organization, from genes to colonies. The evolutionary significance of modularity is considerable, with potential effects including constraining the variation of individual traits, circumventing pleiotropy and canalization, and facilitating the transformation of functional structures. Despite these important consequences, there has been little empirical study of how modularity influences morphological evolution on a macroevolutionary scale. Here, we conduct the first morphometric analysis of modularity and disparity in two clades of placental mammals, Primates and Carnivora, and test if trait integration within modules constrains or facilitates morphological evolution.

Principal Findings

We used both randomization methods and direct comparisons of landmark variance to compare disparity in the six cranial modules identified in previous studies. The cranial base, a highly-integrated module, showed significantly low disparity in Primates and low landmark variance in both Primates and Carnivora. The vault, zygomatic-pterygoid and orbit modules, characterized by low trait integration, displayed significantly high disparity within Carnivora. 14 of 24 results from analyses of disparity show no significant relationship between module integration and morphological disparity. Of the ten significant or marginally significant results, eight support the hypothesis that integration within modules constrains morphological evolution in the placental skull. Only the molar module, a highly-integrated and functionally important module, showed significantly high disparity in Carnivora, in support of the facilitation hypothesis.

Conclusions

This analysis of within-module disparity suggested that strong integration of traits had little influence on morphological evolution over large time scales. However, where significant results were found, the primary effect of strong integration of traits was to constrain morphological variation. Thus, within Primates and Carnivora, there was some support for the hypothesis that integration of traits within cranial modules limits morphological evolution, presumably by limiting the variation of individual traits.     

Phenotypic Integration Without Modularity: Testing Hypotheses About the Distribution of Pleiotropic Quantitative Trait Loci in a Continuous Space

Theories of phenotypic integration have relied heavily on the concept of modularity in order to model the ways in which traits in an organism correlate and covary. Recent investigations suggest that, while some functional and developmental processes may be morphologically and ontogenetically localized, and thus modular in a developmental sense, there is a great deal of overlap among these influences on patterns of integration in the adult form. This can result in blurry boundaries between hypothesized modules constructed to test hypotheses about phenotypic integration. This investigation tests hypotheses about the contribution of pleiotropic quantitative trait loci (QTL) to phenotypic integration in the mouse mandible without using a priori categorical hypotheses about which traits constitute a module. We ask two main questions: (1) Are the effects of pleiotropic QTL localized to highly correlated traits or more spread out among traits than one might expect by chance? (2) Does the pattern of trait influence when all pleiotropic QTL are considered together deviate from what we might expect if QTL affect traits without regard for the correlations among traits? We find that a large proportion of pleiotropic QTL affect traits that are more highly correlated than we expect by chance with the remainder having effects that are distributed as if by chance. Furthermore, the overall distribution of the effects of pleiotropic QTL differs significantly from the null distribution of no association between pleiotropic effects on traits and correlations among traits. The main modular hypothesis used by earlier studies often does not predict the distribution of sets of traits sharing a common QTL. These results suggest that there is a clear tendency for pleiotropic effects of QTL to be localized but that the localization may be best thought of as occurring in a continuous space rather being clustered in discrete modules.     

Multilevel assessment of the Lacertid lizard cranial modularity

Different factors and processes that produce phenotypic variation at the individual, population, or interspecific level can influence or alter the covariance structure among morphological traits. Therefore, studies of the patterns of integration and modularity at multiple levels—static, ontogenetic, and evolutionary, can provide invaluable data on underlying factors and processes that structured morphological variation, directed, or constrained evolutionary changes. Our dataset, consisting of cranium shape data for 14 lizard species from the family Lacertidae, with substantial samples of hatchlings and adults along with their inferred evolutionary relationships, enabled us to assess modularity and morphological integration at all three levels. Five, not mutually exclusive modularity hypotheses of lizard cranium, were tested, and the effects of allometry on intensity and the pattern of integration and modularity were estimated. We used geometric morphometrics to extract symmetric and asymmetric, as well as allometric and nonallometric, components of shape variation. At the static level, firm confirmation of cranial modularity was found for hypotheses which separate anterior and posterior functional compartments of the skull. At the ontogenetic level, two alternative hypotheses (the “anteroposterior” and “neurodermatocranial” hypotheses) of ventral cranial modularity were confirmed. At the evolutionary level, the “neurodermatocranial” hypothesis was confirmed for the ventral cranium, which is in accordance with the pattern observed at the ontogenetic level. The observed pattern of static modularity could be driven by functional demands and can be regarded as adaptive. Ontogenetic modularity and evolutionary modularity show the same developmental origin, indicating conservatism of modularity patterns driven by developmental constraints.     

Testing for environmentally induced bias in phenotypic estimates of natural selection: theory and practice

Measuring natural selection has been a fundamental goal of evolutionary biology for more than a century, and techniques developed in the last 20 yr have provided relatively simple means for biologists to do so. Many of these techniques, however, share a common limitation: when applied to phenotypic data, environmentally induced covariances between traits and fitness can lead to biased estimates of selection and misleading predictions about evolutionary change. Utilizing estimates of breeding values instead of phenotypic data with these methods can eliminate environmentally induced bias, although this approach is more difficult to implement. Despite this potential limitation to phenotypic methods and the availability of a potential solution, little empirical evidence exists on the extent of environmentally induced bias in phenotypic estimates of selection. In this article, we present a method for detecting bias in phenotypic estimates of selection and demonstrate its use with three independent data sets. Nearly 25% of the phenotypic selection gradients estimated from our data are biased by environmental covariances. We find that bias caused by environmental covariances appears mainly to affect quantitative estimates of the strength of selection based on phenotypic data and that the magnitude of these biases is large. As our estimates of selection are based on data from spatially replicated field experiments, we suggest that our findings on the prevalence of bias caused by environmental covariances are likely to be conservative.     

QUANTITATIVE GENETICS OF SHAPE IN CRICKET WINGS: DEVELOPMENTAL INTEGRATION IN A FUNCTIONAL STRUCTURE

The role of developmental and genetic integration for evolution is contentious. One hypothesis states that integration acts as a constraint on evolution, whereas an alternative is that developmental and genetic systems evolve to match the functional modularity of organisms. This study examined a morphological structure, the cricket wing, where developmental and functional modules are discordant, making it possible to distinguish the two alternatives. Wing shape was characterized with geometric morphometrics, quantitative genetic information was extracted using a full‐sibling breeding design, and patterns of developmental integration were inferred from fluctuating asymmetry of wing shape. The patterns of genetic, phenotypic, and developmental integration were clearly similar, but not identical. Heritabilities for different shape variables varied widely, but no shape variables were devoid of genetic variation. Simulated selection for specific shape changes produced predicted responses with marked deflections due to the genetic covariance structure. Three hypotheses of modularity according to the wing structures involved in sound production were inconsistent with the genetic, phenotypic, or developmental covariance structure. Instead, there appears to be strong integration throughout the wing. The hypothesis that genetic and developmental integration evolve to match functional modularity can therefore be rejected for this example.     

Morphological integration and serial homology: A case study of the cranium and anterior vertebrae in salamanders

Serial homology or the repetition of equivalent developmental units and their derivatives is a phenomenon encountered in a variety of organisms, with the vertebrate axial skeleton as one of the most notable examples. Serially homologous structures can be viewed as an appropriate model system for studying morphological integration and modularity, due to the strong impact of development on their covariation. Here, we explored the pattern of morphological integration of the cranium and the first three serially homologous structures (atlas, first, and second trunk vertebrae) in salamandrid salamanders, using micro-CT scanning and three-dimensional geometric morphometrics. We explored the integration between structures at static and evolutionary levels. Effects of allometry on patterns of modularity were also taken into account. At the static level (within species), we analyzed inter-individual variation in shape to detect functional modules and intra-individual variation to detect developmental modules. Significant integration (based on inter-individual variation) among all structures was detected and allometry is shown to be an important integrating factor. The pattern of intra-individual, asymmetric variation indicates statistically significant developmental integration between the cranium and the atlas and between the first two trunk vertebrae. At the evolutionary level (among species), the cranium, atlas, and trunk vertebrae separate as different modules. Our results show that morphological integration at the evolutionary level coincides with morphological and functional differentiation of the axial skeleton, allowing the more or less independent evolutionary changes of the cranial skeleton and the vertebral column, regardless of the relatively strong integration at the static level. The observed patterns of morphological integration differ across levels, indicating different impacts of developmental and phylogenetic constraints and functional demands.     

Drosophila wing modularity revisited through a quantitative genetic approach

To predict the response of complex morphological structures to selection it is necessary to know how the covariation among its different parts is organized. Two key features of covariation are modularity and integration. The Drosophila wing is currently considered a fully integrated structure. Here, we study the patterns of integration of the Drosophila wing and test the hypothesis of the wing being divided into two modules along the proximo‐distal axis, as suggested by developmental, biomechanical, and evolutionary evidence. To achieve these goals we perform a multilevel analysis of covariation combining the techniques of geometric morphometrics and quantitative genetics. Our results indicate that the Drosophila wing is indeed organized into two main modules, the wing base and the wing blade. The patterns of integration and modularity were highly concordant at the phenotypic, genetic, environmental, and developmental levels. Besides, we found that modularity at the developmental level was considerably higher than modularity at other levels, suggesting that in the Drosophila wing direct developmental interactions are major contributors to total phenotypic shape variation. We propose that the precise time at which covariance‐generating developmental processes occur and/or the magnitude of variation that they produce favor proximo‐distal, rather than anterior‐posterior, modularity in the Drosophila wing.     

In search of morphological modules: a systematic review

Morphological modularity arises in complex living beings due to a semi‐independent inheritance, development, and function of body parts. Modularity helps us to understand the evolvability and plasticity of organismal form, and how morphological variation is structured during evolution and development. For this reason, delimiting morphological modules and establishing the factors involved in their origins is a lively field of inquiry in biology today. Although it is thought that modularity is pervasive in all living beings, actually we do not know how often modularity is present in different morphological systems. We also do not know whether some methodological approaches tend to reveal modular patterns more easily than others, or whether some factors are more related to the formation of modules or the integration of the whole phenotype. This systematic review seeks to answer these type of questions through an examination of research investigating morphological modularity from 1958 to present. More than 200 original research articles were gathered in order to reach a quantitative appraisal on what is studied, how it is studied, and how the results are explained. The results reveal an heterogeneous picture, where some taxa, systems, and approaches are over‐studied, while others receive minor attention. Thus, this review points out various trends and gaps in the study of morphological modularity, offering a broad picture of current knowledge and where we can direct future research efforts.     

Evolution of phenotypic integration in Brassica (Brassicaceae)

Phenotypic integration is a necessary characteristic of living organisms that results from genetic, developmental, and functional relationships among traits. The nature of these relationships can be influenced by the environment. We examined patterns of phenotypic integration of six species of rapid cycling Brassica and of Raphanus sativus within a phylogenetic context. Specifically, we tested the hypothesis that hybrid species show intermediate levels of integration in morphological and life-history characters compared to their putative parentals. We used matrix correlation tests to examine if cytogenetic relationships or ecological similarities among species partially explained the patterns of phenotypic integration. There was a significant negative relationship between the ecological and cytogenetic matrices, suggesting that more closely related species were ecologically dissimilar. However, neither ecological nor cytogenetic matrices significantly explained differences among species in the pattern of their phenotypic correlations. Set correlation analysis indicated that important traits within the modules and the strength of the correlations within modules differed across species. We also found that there were a greater number of significant correlations between modules than within modules. Hybrid species were more integrated (had greater number of significant trait correlations) than either of their parents, both within and between modules. However, univariate analyses of character means of the hybrid species were not significantly different from the combined mean of their putative parents for 5, 6, or 7 of the 11 phenotypic characters (for Brassica napus, B. juncea and B. carinata, respectively); for the remaining characters, the hybrids were more similar to one of the parents.     

A comparison of methods to calculate a total merit index using stochastic simulation

Background

Modern dairy cattle breeding goals include several production and more and more functional traits. Estimated breeding values (EBV) that are combined in the total merit index usually come from single-trait models or from multivariate models for groups of traits. In most cases, a multivariate animal model based on phenotypic data for all traits is not feasible and approximate methods based on selection index theory are applied to derive the total merit index. Therefore, the objective of this study was to compare a full multitrait animal model with two approximate multitrait models and a selection index approach based on simulated data.

Methods

Three production and two functional traits were simulated to mimic the national Austrian Brown Swiss population. The reference method for derivation of the total merit index was a multitrait evaluation based on all phenotypic data. Two of the approximate methods were variations of an approximate multitrait model that used either yield deviations or de-regressed breeding values. The final method was an adaptation of the selection index method that is used in routine evaluations in Austria and Germany. Three scenarios with respect to residual covariances were set up: residual covariances were equal to zero, or half of or equal to the genetic covariances.

Results

Results of both approximate multitrait models were very close to those of the reference method, with rank correlations of 1. Both methods were nearly unbiased. Rank correlations for the selection index method showed good results when residual covariances were zero but correlations with the reference method decreased when residual covariances were large. Furthermore, EBV were biased when residual covariances were high.

Conclusions

We applied an approximate multitrait two-step procedure to yield deviations and de-regressed breeding values, which led to nearly unbiased results. De-regressed breeding values gave even slightly better results. Our results confirmed that ignoring residual covariances when a selection index approach is applied leads to remarkable bias. This could be relevant in terms of selection accuracy. Our findings suggest that the approximate multitrait approach applied to de-regressed breeding values can be used in routine genetic evaluation.